Sequencing of Erns gene of a live attenuated classical swine fever cell culture vaccine virus and its comparison with back passages and other CSFV strains

Phylogenetic relationship of the CSF cell culture vaccine virus has also been established with other known CSF reference vaccine viruses, lapinized as well as cell culture adapted strains.

Original Research Article https://doi.org/10.20546/ijcmas.2020.908.104

Sequencing of Erns Gene of a Live Attenuated Classical Swine Fever Cell Culture Vaccine Virus and its Comparison with Back Passages and Other

CSFV Strains

B Ompreethi, M Manu, R Pachauri, V Upamanyu, A K Tiwari and P Dhar *

Division of Biological Standardization, ICAR-Indian Veterinary Research Institute,

Izatnagar, India

*Corresponding author

A B S T R A C T

Introduction

Classical swine fever (CSF) is a highly fatal

disease of pigs caused by classical swine

fever virus belonging to the genus Pestivirus

that also includes bovine viral diarrhea virus

(BVDV) and border disease virus (BDV) It

is a small enveloped virus with a

single-stranded positive sense RNA of size of

12.3-kb with the genome structure of

5'UTR-Npro-C-Erns

-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3'UTR (Rice, 1996) Among the

11-12 viral proteins, E2 is the major glycoprotein anchored in the envelope and consists of 373 amino acids having molecular weight of 51-55 kDa The Erns is the second protein of importance, consisting of 227 amino acids (residue 268 to 494) with molecular weight of 41-44 kDaand is located

at nucleotide positions 1178 to 1858of the genome (681 bases) downstream of the C

protein (Zhang et al., 2011, Leifer et al.,

2010) The Erns protein isloosely attached to the envelope and has been known to be

ISSN: 2319-7706 Volume 9 Number 8 (2020)

Journal homepage: http://www.ijcmas.com

The Erns gene (681 bases) of a live attenuated classical swine fever (CSF) cell culture Indian vaccine virus (IVRI-CSF-BS)was sequenced and had only three nucleotide changes compared to its parental virulent virus at passage 6 in cell culture The vaccine virus had Thymine at 151 and Guanine at 184 and 638 positions instead of Cytosine and Adenine at respective places in p6 virus Out of these three mutations, nucleotide changes at 184 and

638 positions resulted in amino acid changes from Lysine to Glutamic acid and Arginine, respectively The Erns sequences of the vaccine virus were same as in the back passages up

to passage 20 Further down at passage 15, the sequences were same except for Adenine at the 638 position, like it was in the p6 virus Overall p15 had one amino acid change (Glutamic acid) and from p20 onwards, the viruses had two amino acid changes (Glutamic acid and Arginine) These changes were however not linked to virus attenuation, as the p20 virus produced fatal CSF infection in susceptible piglets Additionally, the vaccine virus was phylogenetically more related to other CSF cell culture vaccine viruses derived from virulent CSF viruses than the lapinized vaccine viruses

K e y w o r d s

Classical swine

fever virus, Indian,

Cell culture

vaccine, Erns gene,

Mutation

Accepted:

10 July 2020

Available Online:

10 August 2020

Article Info

Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 960-968

responsible for virulence of the CSF viruses

Mutations at certain locations of the Erns gene

have been attributed for attenuation of the

virus (Meyers et al., 1999; Sainz et al., 2008;

Tews et al., 2009) Since we have both a CSF

cell culture vaccine virus (IVRI-CSF-BS) as

well as its parental virulent virus in our

laboratory, we investigated the Erns genes of

these viruses as well as some passages in

between, for any evidence of change of

sequences and its effect on virus attenuation

Phylogenetic relationship of the CSF cell

culture vaccine virus has also been

established with other known CSF reference

vaccine viruses, lapinized as well as cell

culture adapted strains

Materials and Methods

Virus

A live attenuated CSF cell culture Indian

vaccine virus (IVRI-CSF-BS) and some of its

back passages such as passage numbers 15,

20, 33, 42, and 51 available in the laboratory

were used for RNA extraction, PCR

amplification and Sanger sequencing of the

Erns gene

Primers

Primers were designed based on the available

sequence of cell culture adapted CSF

challenge virus at passage 6 (Accession

No.MG599478) and other CSF viruses Oligo

Analyzer software was used for primer

designing and were verified by Primer Blast

(Table 1)

RNA isolation

Isolation of RNA from the live attenuated

CSF vaccine virus (IVRI-CSF-BS) and its

back passages were done directly from the

virus samples, stored either in freeze dried or

in liquid form at -20C since 2017 Freeze

dried virus samples were reconstituted directly in 1000 µl of Tri-reagent (Sigma Cat

#T9424) and incubated at room temperature (RT) for 5 minutes Two hundred µl of chloroform was added and incubated at RT for 15 min and centrifuged at 12000 rpm for

15 min at 4ºC Aqueous phase was collected and incubated with equal volume of isopropanol at RT for 10 min and centrifuged

at 12000 rpm for 10 min at 4ºC Supernatant was discarded and the pellet was washed in

500 µl of 70% ethanol by vortexing for 8 seconds The solution was then centrifuged at

7500 rpm for 5 min at 4ºC Finally, the supernatant was discarded and the pellet was air dried for 10 minutes The RNA pellet was dissolved in 11 µl of nuclease free water (Thermo Scientific Cat#R0582) containing 20 units of RNase inhibitor (Ribolock, ThermoScientific Cat # R0582) and stored at -80ºCuntil used

For RNA extraction from the liquid viruses,

750 µl of TRI-reagent was added to 250 µl of the liquid viruses and thereafter followed the same steps as done for freeze dried viruses

Reverse Transcription-PCR

The RNAs were reverse transcribed to synthesize 20µl of complementary DNA (cDNA) using a commercial kit (Thermo Scientific Revertaid First Strand cDNA synthesis kit, Cat # K1632) as per the manufacturers protocol and were stored at -20ºC until used

Since the virus samples were almost two years old (stored since 2017), the cDNAs derived from these were first checked by a Taq polymerase PCR for amplification of the

Erns gene (763bp), before actually amplifying

it using a proof reading KOD polymerase (Merck Cat #71842)for sequencing purpose Briefly, 2.5l cDNAs were added to the PCR reaction mix containing 10 pmol of forward

and reverse primers (CSFV-10-Erns and

CSFV-Erns-34) (Table 1), 0.25units of Taq

Polymerase (ThermoScientific Cat#EP0404),

1.5mM MgCl2, 2mM dNTPs, in 1x Taq

polymerase buffer and total reaction volume

was made to 25 l with nuclease free water

The PCR reactions were done in Eppendorf

Master Cycler PCR machine The reactions

were subjected to initial denaturation of the

cDNAs at 95ºC for 5 min, followed by 34

cycles of denaturation, annealing and

extension at 95ºC for 30 sec, 48ºC for 30 sec

and 72ºC, 60 sec respectively, followed by

final extension at 72ºC for 5 minutes Ten l

of the PCR reactions were mixed in 2l of 6x

loading dye and run in a freshly prepared

1.2% agarose gel in 0.5xTBE buffer

containing 2l Red safe dye (Intron Cat #

21141) for 45 min at 100V power and

checked for 763 bp size amplicon under U.V

Transilluminator (Gel Doc XR,BioRad)

Once the cDNAs were checked, these were

used for PCR amplification of the Erns gene

using a KOD hot start master mix, that

contains a proof reading polymerase Briefly,

2l cDNAs were added to 50µl reaction mix

containing 20 pmol of each of the primers, 1x

KOD master mix and the final volume is

made with nuclease free water The PCR

steps consisted of denaturation at 95ºC for 2

min followed by 30 cycles of denaturation,

annealing and extension at 95ºCx20 sec,

48ºCx10 sec and 70ºCx15 sec, respectively

and final extension at 70ºC for 10 seconds

The PCR products were detected in 1.2%

agarose gel and further purified by gel

extraction using a gel extraction kit (GCC

Biotech Cat# G4628A)

Sequencing of the E rns gene of the back

passages

The 763bp of the Erns amplicon of the CSF

vaccine virus and its back passages (passage

numbers 15, 20, 33, 42 and 51)(Fig 1) were

sequenced by Sanger's sequencing (Eurofins India Pvt ltd)using internal primers

CSFV-Erns-72and CSFV-Erns-380 (Table 1).The sequences of only the 681 bp of the Erns genes were aligned with the other CSFV sequences

in NCBI blast analysis using MEGA X software Phylogenetic analysis of the sequences was done by CLUSTAL W program

Results and Discussion

Sequencing of E rns gene of CSF cell culture vaccine and its back passages

The 681bp nucleotide sequence of the Erns gene of the live attenuated CSF cell culture vaccine of Indian origin (IVRI-CSF-BS) has been submitted in GenBank (Accession number MT424777) Upon analysis of the sequence, we observed only three nucleotide changes in the Erns gene in the vaccine virus compared to its parental virulent virus

(Badasara et al., 2017) at passage 6 in cell

culture The three mutations were CytosineThymine at 151 and Adenine

Guanine at 184 and 638 positions The first two nucleotide substitutions of CT and A

Gat 151 and 184 positions respectively were detected from passage 15 (Fig 2a) and the third mutation of AG at 638 position was detected from passage 20 onwards (Fig 2b) The mutation of CTat 151 position was a synonymous mutation without any change in amino acid sequence, and this has been observed only in the Indian vaccine virus (IVRI-CSF-BS) compared to other CSF vaccine of virulent viruses including the parental virulent virus at passage 6 (Fig 3).The second nucleotide substitution from

AG at position184 in the same passage 15 virus resulted in change of amino acid from Lysine to Glutamic acid at amino acid position 62 Subsequently, in passage 20 onwards, another similar nucleotide substitution from AG was again observed at

Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 960-968

638 position of the Erns gene and this resulted

in a change of amino acid from Lysine to

Arginine at 213 position of Erns protein No

further changes in the Erns gene were observed

in subsequent passages Thus, the Erns of the

CSF virus at passage15 had only one amino

acid change and from passage 20 onwards till

the vaccine stage, the viruses had two amino

acid changes Our findings are in agreement

in respect to a PK-15 adapted French CSF

Thiverval vaccine strain (accession no

EU490425) with its parental virulent Alfort

187 strain, where also only three nucleotide

changes were observed between the two

viruses at 348, 638 and 669 positions with

only one amino acid change from

Lysine-to-Arginine at 213 position of Erns protein (Fan

et al., 2008) Similarly, while comparing the

Erns sequences of a guinea pig adapted GPE- vaccine strain (Accession no D49533) with its ancestral virulent ALD strain, six mutations were observed at 187, 284, 318,

320, 321 and 435 positions with only three amino acid changes from Glycine-to-Arginine

at 63 position, Asparagine-to-Serine at 95 position and Alanine-to-Aspartic acid at 107 amino acid positions in Erns protein (Ishikawa

et al., 1995) Thus, it may be possible that the

mutations as accumulated in the Erns gene, actually depend on the type of the cell culture used for passaging of the virulent parental virus

Table.1 Primers used in the study

1

CSFV-10-E rns -F

protein gene upstream of the

E rns gene

amplification of the

E rns gene (product size of 763 bp) CSFV- E rns -34-R rttagtgtaccatatgtacc 1337-1318 in the

E1 protein gene downstream of the E rns gene

20

2 CSFV-E rns -72F gtcagcagaagtttgcatg 676-694 in E rns 19 For use as internal

primers for sequencing CSFV- E rns -380R cctgagtgaccacattgac 985-967 in E rns 19

amplicon was generated for the back passages such as P15, P20, P33, P42, P51 (Lanes 1, 2, 3, 4

and 5 respectively) and the CSF vaccine virus (Lane 6); M – 100bp DNA ladder; Lane 7- No

amplification in the control The 763 bp amplicon was sequenced and only 681 bp sequences of

the complete Erns gene was used in sequence analysis

763bp

Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 960-968

7) with its back passages (Sl No 2 – 6) and the parental virulent virus at P-6 in cell culture (Sl

No 1) are shown Two mutations of C151T and A184G were observed from P15 onwards

7) with its back passages (Sl No 2 – 6) and the parental virulent virus at P-6 in cell culture (Sl

No 1) are shown Only one mutation of A638G was observed from P20 onwards [Nucleotides

of the E rns of the CSF vaccine virus other than at the positions 151,184 and 638 were similar to

that of the parental virulent virus at p6 in cell culture]

the CSF vaccine virus (IVRI-CSF-BS) compared to its parental virulent virus at passage 6 in cell culture (Serial No 2) and other 23 CSF vaccine or virulent field viruses which had Cytosine (C)

in the same position

84

151

638

151

151

Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 960-968

CSF cell culture vaccine virus (IVRI-CSF-BS) has 92.8 to 94.9% homology with other cell culture vaccines whereas 90 to 91.5% with the Lapinized vaccines and 81.4 to 94.9% with the

field isolates

culture vaccine virus (IVRI-CSF-BS) is in the same cluster along with its parental virulent virus

at passage 6 in cell culture (MG599478) and the original Indian virulent virus (MK405703.1) from which the vaccine virus was derived These viruses are more closely related to the other

cell cultures vaccines than the lapinized vaccines

Fig.6 Rectal temperature of pigs inoculated with P20 virus Biphasic temperature reactions were

observed in both the pigs (916 and 919) with peak temperature 104.5° F from 7 to 11 days post inoculation Both the pigs had died of CSF infection after showing the clinical symptoms of CSF and had marked leucopenia (1400 and 2950 cells/cu mm) The passage 20 virus was a hot virus although two amino acid changes have been accumulated in the Erns sequence compared to its

parental virulent virus

Fig.7 Detection of CSFV genome by RT-PCR in the pig blood collected at viraemia stage (10

dpi) after inoculation of the P20 virus A 421 bp amplicon was detected in the blood of pig no

919 (Lane-1); M-100 bp DNA ladder; Lane 2- No amplification in the negative control

analysis with CSFV reference vaccine

strains

The live attenuated CSF cell culture Indian

vaccine virus (IVRI-CSF-BS) has been

developed recently and its molecular

characterization has not yet done We

compared the Erns gene of the Indian cell culture vaccine virus with that of the lapinized vaccines (such as Chinese C strain, Riems strain, Russian KC strain, Indian Lapinized strain, Swedish ROVAC strain, etc) as well as other cell culture vaccines derived from virulent field isolates (such as Thiverval strain, GPE- strain, LOM strain, etc) (Fig 4)

421bp

500 bp 400bp 300bp 200bp 100bp 1000bp

1 M 2

Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 960-968

Based on the sequence analysis of the Erns

genes of these viruses, we observed that our

cell culture Indian vaccine virus has more

sequence homology (92.8 to 94.9%) with

other live attenuated cell culture vaccines than

the lapinized vaccines (90 to 91.5%)

including the widely known Chinese C strain

(91.5%) and the Indian lapinized vaccine

(91.3%) Phylogenetic analysis of the

Erns genes also revealed the same Our vaccine

virus (IVRI-CSF-BS) is more closely related

to other CSF cell culture adapted viruses than

the lapinized vaccine strains (Fig

5).Additionally, the CSF Indian vaccine virus

(IVRI-CSF-BS) and its parental virulent virus

have been found to stand out as a separate

group within the CSF cell culture vaccine

viruses

Correlation of mutations in the E rns gene of

CSFV with virus attenuation

The nucleotide sequences of the Erns genes of

the Indian vaccine virus and the back

passages revealed that the virus had two

changes in amino acid of the Erns protein from

passage 20 onwards Since Erns in known to

be a virulence determinant of CSF viruses and

mutations in this gene is known to cause virus

attenuation (Meyers et al., 1999; Sainz et al.,

2008; Tews et al., 2009), we attempted to

look for the virus attenuation of the passage

20 virus in vivo However, we observed that

these amino acid changes in the Erns are not

linked to virus attenuation as the 20th passage

virus at a dose of 105.5 TCID50produced CSF

infections in two susceptible piglets which

ultimately succumbed to the infection in 17

days The piglets had the CSF symptoms

starting from anorexia, depression to fever up

to 104.5°F (Fig 6), skin rashes in the

extremities, emaciation, paralysis of

hindquarters, respiratory distress and diarrhea

before finally dying due to the disease Both

the animals had marked leukopenia such as

1400 cells/cu.mm in one and 2950 cell/cu.mm

in the second one, which is most pathognomic for CSF infection Postmortem examination

of these animals were also suggestive of CSF infection such as pinpoint hemorrhages on the kidneys (turkey egg appearance), hemorrhages on the tonsils, necrotic lesions in the intestine and necrosis of mesenteric and

other lymph glands (Badasara et al., 2017)

The blood collected at viraemia stage were also positive in CSF specific PCR (Fig 7) Thus, we confirmed that the passage 20 virus was still a hot virus and the two amino acid changes had actually no effect on the virus

attenuation invivo Our study is in agreement

with an earlier study on the virulent Brescia strain, in which a Serine-to-Arginine at 209 position did not reduce virulence in pigs (Van

Gennip et al., 2004) although this position is

known to be responsible for heparin sulfate

receptor binding into the cells (Hulst et al.,

2000)

In conclusion there was not much change in the Erns gene between the virulent and the

vaccine virus The unique change i.e., CT

at 151 position can be used to identify the virus as it is observed only in the Indian vaccine virus (IVRI-CSF-BS) and can be attributed as a signature nucleotide change The little changes occurred were however not related to virus attenuation

Acknowledgement

The authors would like to acknowledge the Director, ICAR-IVRI, Joint Director (Academic), ICAR-IVRI Deemed University and Head, Division of Biological Standardization, ICAR-IVRI for facilities to carry out present work The authors would also like to thank Dr Y.P.S Malik, Principal Scientist of the Division and the students and Research Assistant of his laboratory for providing the facility of PCR machine and also providing some necessary chemicals used

in the study

References

Badasara, S.K., Mohan, M., Sah, V., Kumari,

P., Upamanyu, V., Dhar, P., Tiwari,

A.K., Chander, V and Gupta, V.K

(2017) Replacement of Animal Model

for Propagation of Classical Swine Fever

Challenge Virus by Adaption in the

Research, 7(3): 581-586

Fan, Y., Zhao, Q., Zhao, Y., Wang, Q., Ning, Y

and Zhang, Z (2008) Complete genome

sequence of attenuated low-temperature

Thiverval strain of classical swine fever

virus Virus genes, 36(3): 531-538

Hulst, M.M., Van Gennip, H.G.P and

Moormann, R.J.M., (2000) Passage of

classical swine fever virus in cultured

swine kidney cells selects virus variants

that bind to heparan sulfate due to a

single amino acid change in envelope

protein Erns Journal of virology, 74(20):

9553-9561

Ishikawa, K., Nagai, H., Katayama, K., Tsutsui,

M., Tanabayashi, K., Takeuchi, K.,

Hishiyama, M., Saitoh, A., Takagi, M.,

Gotoh, K and Muramatsu, M (1995)

Comparison of the entire nucleotide and

deduced amino acid sequences of the

attenuated hog cholera vaccine strain

GPE− and the wild-type parental strain

1385-1391

Leifer, I., Hoffmann, B., Hoper, D., Rasmussen,

T.B., Blome, S., Strebelow, G.,

Horeth-Bontgen, D., Staubach, C and Beer, M

(2010) Molecular epidemiology of

current classical swine fever virus

Virology, 91(11): 2687-2697

Meyers, G., Saalmüller, A and Büttner, M (1999) Mutations abrogating the RNase activity in glycoprotein Erns of the pestivirus classical swine fever virus lead

Virology, 73(12): 10224-10235

Rice, C.M (1996) Flaviviridae: the viruses and their replication, In: Knipe, B.N F.D.M.,

Virology, Third ed Lippincott Raven, Philadelphia: 931–959

Sainz, I.F., Holinka, L.G., Lu, Z., Risatti, G.R and Borca, M.V (2008) Removal of a N-linked glycosylation site of classical swine fever virus strain Brescia Erns

swine Virology, 370(1): 122-129

Tews, B.A., Schürmann, E.M and Meyers, G (2009) Mutation of cysteine 171 of

homodimer formation and leads to attenuation of classical swine fever

virus Journal of Virology, 83(10):

4823-4834

Van Gennip, H.G.P., Vlot, A.C., Hulst, M.M.,

De Smit, A.J and Moormann, R.J.M., (2004) Determinants of virulence of classical swine fever virus strain

Brescia Journal of Virology, 78(16):

8812-8823

Zhang H., Cao H.W., Wu Z.J and Cui Y.D

Characterization of Classical Swine

FeverVirus (CSFV) Israel Journal of

Veterinary Medicine, 66 (3):89-95

How to cite this article:

Ompreethi, B., M Manu, R Pachauri, V Upamanyu, A K Tiwari and Dhar, P 2020 Sequencing of Erns Gene of a Live Attenuated Classical Swine Fever Cell Culture Vaccine Virus and its Comparison with Back Passages and Other CSFV Strains

Int.J.Curr.Microbiol.App.Sci 9(08): 960-968 doi: https://doi.org/10.20546/ijcmas.2020.908.104